The present invention relates to techniques for detection and identification of biological contaminants in vegetable foodstuffs. More particularly, the present invention involves the detection of insects or insect parts in whole or milled grain.
The detection of insects in stored grain and the quantitation of insect parts present in milled grain products represents a serious and continuing problem for the cereal industry. Present methods of detection primarily involve visual inspection [usually microscopic] (1, 2) and x-ray analysis (3) which require trained personnel and are time-consuming, difficult to standardize and expensive.
A variety of approaches have been used in the past to attempt development of efficient assay procedures, but none of these has proven particularly satisfactory for routine testing. These methods have included the use of nuclear magnetic resonance (4, 5, 6), sound amplification (7), infrared spectrometry (8), and chemical techniques (9). These techniques have tended to be too expensive, technically difficult and also difficult to quantify and identify the specific infestation detected. For example, sound amplification techniques (10-12) show promise for the detection of live insects in grain but provide little quantitative analysis, and are unable to detect eggs, dead insects or insect part contamination. The preferred characteristics of assays for insect contamination are as follows: it should be highly specific, be very sensitive, rapid, and inexpensive. Moreover, it ideally should be employable by persons having minimal training, particularly in non-laboratory settings, e.g., at grain elevator and mill sites.
The present invention involves approaches to the detection of insect contamination of foodstuffs such as grain, beans (e.g., coffee), spices or even viable crops (e.g., corn, etc.). These approaches involve the detection or discrimination of biological substances specific to insects and usually absent in pure foodstuffs. One such approach is an assay for the insect exoskeleton material, chitin. This test is particularly suited to the detection of live or dead specimens of the adult stages of insects and for insect parts. Another type of assay uses an immunological approach for the detection and quantitation of an insect-specific protein such as the insect muscle protein, myosin or components thereof, for example. This type of test is well suited for detecting all stages of insect development from egg to adult, whether live or dead. It should also provide an assay system that correlates well to the current insect fragment assay and whole insect analysis (eggs to adults).
Chitin is the major structural material of insect exoskeletons. This material is a beta-[1-4]-homopolymer of N-acetylglucosamine, as shown in FIG. 1. Chitin is not found in higher plants. Because of the relatively large amounts of chitin associated with the pupal and adult stages of insects, sensitive biochemical tests for chitin should provide a good means of assaying for the presence of both live and dead insect remains in plant material. Chitin may also be found in certain molds and fungi which could be an important consideration in some circumstances.
Very sensitive and rapid procedures are available for assaying for chitin. Such tests can be modified for successful determination of grain or grain product insect infestation. The most sensitive test procedures for chitin involve hydrolysis of the chitin to N-acetylglucosamine and subsequent assay for the latter compound. Several chemical degradation procedures for chitin hydrolysis have been tested and several N-acetylglucosamine tests evaluated for their suitability for grain assays. One sensitive and reliable assay technique described herein uses an alkaline hydrolysis procedure and has a current detection limit of approximately 1.times.10.sup.-9 moles of N-acetylglucosamine (NAG). The average grain weevil contains approximately 1.12.times.10.sup.-6 moles of NAG, which is 1000 times more NAG than is needed for a positive test. The major potential disadvantage of this assay is that it is rather lengthy and requires the use of harsh chemicals. It is clear that the procedures described herein offer a very sensitive means for grain analysis. The further adaptation of chitin assays for low cost and rapid analysis is an object of the present invention.
Immunological assays have found widespread use in clinical diagnostic settings (13), and are also becoming available for in home use, particularly for the early detection of pregnancy (14). The vast potential of these procedures for non-medical purposes has been the subject of increasing study over the last few years. Nowhere is this more evident than in the explosion of commercial uses being developed for the enzyme linked immunosorbent assays (ELISA), which are rapidly becoming standard procedure in a variety of settings (15).
In order to develop an optimal immunological assay for an insect contamination of foodstuffs, antibodies are required which are directed against an insect-specific antigen, preferably protein, likely to be present in any life stage of the contaminating insect or in insect remains. Necessarily, the antisera should not cross-react with any plant material present. For an immunoassay with broad insect-specificity, it would also be preferable to use as an antigen, a protein that is very slowly evolving. Antibodies directed against such an insect-specific protein of one species would cross-react with the same protein in a wide variety of insect species. One such protein is the insect muscle protein myosin. Myosin and components thereof are ubiquitous in insects. Myosin is present in large quantities in adult insect tissue, and is also present in appreciable quantities in other life stages. Finally, myosin is a very slowly evolving protein (16). To develop an immunoassay specific for a particular species of insect contamination, antibodies having a unique species-specificity could be prepared and used. For example, polyclonal or monoclonal antibody could be developed which is directed toward an antigen specifically characterizing a single insect species. Alternatively, polyclonal antibodies, monoclonal antibodies or mixtures thereof could be developed which recognize an epitope of an insect antigen which is common to several or all insect species of interest. Such a broadly specific monoclonal antibody could also serve in reproducible insect assays. Monoclonal antibodies provide a dependable source of identical antibodies.